Method for preparing protein cage, and in situ method for preparing hydrophobic additive-supported core-shell structured polymer-protein particles

ABSTRACT

The present invention relates to a method for preparing a protein cage which comprises: a 1 st  step of preparing an amphiphilic polymer comprising a 1 st  hydrophobic polymer and a 1 st  hydrophilic functional group; a 2 nd  step of preparing a hydrophilic protein comprising a 2 nd  functional group binding to the 1 st  functional group; a 3 rd  step of forming an amphiphilic polymer-protein hybrid by the binding of the 1 st  functional group and the 2 nd  functional group, and forming core-shell structured particles comprising a protein shell and an amphiphilic polymer core by the self-assembly of the amphiphilic polymer in a hydrophilic solvent; and a fourth step of removing some or all of the hydrophobic polymer of the core part from the core-shell structured particles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase application under 35 U.S.C. §371 ofInternational Application No. PCT/KR2013/009120 filed 11 Oct. 2013,which claims priority to Korean Patent Application No. 10-2013-0031128filed 22 Mar. 2013. The entire contents of each of the above-referenceddisclosures is specifically incorporated by reference herein withoutdisclaimer.

TECHNICAL FIELD

The present invention relates to a method for preparing a protein cage,a protein cage prepared by the method, an in situ method for preparinghydrophobic additive-loaded core-shell structured polymer-proteinparticles, core-shell structured polymer-protein particles prepared bythe method, and use thereof.

BACKGROUND ART

A drug delivery system (DDS) is a high-value key technology providingeconomic gains comparable to the development of new drugs and having ahigh potential of success, and it pursues an efficient medication,thereby improving the quality of patient treatment. The technique ofsolubilizing poorly soluble drugs, which belongs to a technique forpromoting the absorption of a drug, one of the key techniques in thedrug delivery system, is considered as the most reasonable way to reducethe development costs of new drug substances and at the same timeincrease the value of medicines currently on the market.

Meanwhile, nanocapsule technology can load the component of interestinto a nano-sized capsule and then release it at a desired rate in adesired place. Capsule technology has been studied for a long time. Dueto the limits in capsule size and material, technology developmentthereof has progressed slowly, but has recently been newly spotlightedthrough the development of nanocapsules integrated with nanotechnology.Such nanocapsule technology is applicable to a variety of fieldsincluding fine chemicals, medicaments, cosmetics, electronics, etc.depending on the development process of the capsule material and thetype of substance to be loaded inside the capsule. In particular, in thefields of medicaments, cosmetics, etc., the nanocapsule has thepotential to be variously utilized in targeted cancer therapy, drugdelivery, transdermal absorption of cosmetics, imaging, etc. However,there are disadvantages in that the process of making a nanocapsule iscomplicated and a separate mold for forming the capsule is needed.

Affinity chromatography is a protein separation method using theaffinity between a protein and a ligand (chemicals, amines, amino acids,peptides, proteins) by immobilizing the ligand having a specificinteraction with the protein to be separated on a carrier. It is aselective separation method utilizing the specificity of the targetprotein among a variety of proteins in biological systems and has beenwidely used for separation and purification of fusion proteins andantibodies. In particular, IMAC (immobilized-metal affinitychromatography) is a method for purifying a protein having an affinityfor a transition metal such as Ni²⁺, Co²⁺, Zn²⁺, etc., using a resin asa carrier, in which a ligand is coordinated to the transition metal. Themetal ions such as Ni²⁺, Co²⁺, etc. have been reported to have auniquely high affinity for the histidine tag, and a typical example ofsuch a resin includes Ni-NTA. Nitrilotriacetic acid (NTA) is a metalchelator that forms a complex with a metal ion, and Ni-NTA forms acoordinate bond with the imidazole ring of histidine.

DISCLOSURE OF THE INVENTION Technical Problem

While researching a nanoencapsulation method, the present inventors haveprepared polymer nanoparticles directly coated with a protein without aseparate template, in view of the affinity chromatography used forprotein purification. The inventors have also confirmed that it ispossible to prepare a protein cage by removing some or all of thepolymer from the protein-coated polymer nanoparticle. Furthermore, theinventors have confirmed that a hydrophobic additive can be loadedinside the protein-coated polymer nanoparticles simultaneously with theformation of the nanoparticles by a one-pot encapsulation method, andthus completed the present invention.

Technical Solution

An object of the present invention is to provide a new protein cage; amethod of preparing the same; an in situ method for preparinghydrophobic additive-loaded core-shell structured polymer-proteinparticles; hydrophobic additive-loaded core-shell structuredpolymer-protein particles prepared by the method; and drug deliverysystems, cosmetic compositions, compositions for imaging, artificialvaccines, and biosensors using the same.

Advantageous Effects

In the protein cage according to the present invention, the hydrophobicpolymer used for forming a core is not particularly limited, and it ispossible to introduce a variety of coating proteins since the proteincoating shell can be formed through a binding between the 1^(st) and the2^(nd) functional groups.

Also, using a one-pot reaction without a separate mold, an amphiphilicpolymer-protein hybrid is formed through the binding between the 1^(st)and the 2^(nd) functional groups, and the hybrid can be self-assembledin a hydrophilic solvent to form the core-shell structured particles.And then, some or all of the hydrophobic polymer in the core part can beremoved from the core-shell structured particle to form the proteincage, and this protein cage may be utilized as a protein device in awide variety of fields including cosmetics, medicines, food, healthcare,etc. since various additives such as pharmacologically activesubstances, cosmetic materials, contrast agents, etc. can be loadedinside the protein cage thus formed.

In addition, the manufacturing process is very simple, and it is easy tocontrol the size of core-shell particles, and thus the process can beeffectively utilized in a variety of applications.

Furthermore, the protein cage according to the present invention may besynthesized in a well-defined structure by a simple process, and theexistence of few constraints on the selection and introduction ofproteins makes the cage available in various ways.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram according to one embodiment of the presentinvention showing (a) the formation of a polymer-protein hybrid throughthe binding of X and Y, both of which are functional groups specificallybound to each other, (b) the self-assembly of the polymer-proteinhybrid, and (c) the formation of a polymer-protein hybrid throughvarious bindings.

FIG. 2 is a schematic diagram of a protein cage which can be preparedaccording to one embodiment of the present invention.

FIG. 3 is a diagram illustrating an artificial vaccine which can beprepared according to one embodiment of the present invention.

FIG. 4A illustrates the synthetic mechanism of the polymer whereinNi-NTA is bound to its terminal according to Preparation Example 1.

FIG. 4B is a schematic view illustrating a coordinate bond between thepolymer comprising Ni-NTA at its terminal and the histidine-taggedprotein according to one embodiment of the present invention.

FIG. 4C is a schematic diagram illustrating the formation of Nilered-loaded protein-coated polymer nanoparticles according to oneembodiment of the present invention.

FIG. 6 shows TEM analysis results (A, B) and DLS data (C) of theprotein-coated polymer nanoparticles prepared in Example 1.

FIG. 5 shows TEM analysis results (top) and DLS data (bottom) of theprotein-coated polymer nanoparticles prepared in Example 2.

FIG. 7 shows the size and shape of the polymer-protein particlesprepared in Example 3.

FIG. 8 is DLS data and TEM images showing the size and shape of thepolymer-protein (enzyme) particles prepared in Example 4.

FIG. 9 shows the emission spectrum of the protein-coated polymernanoparticles prepared in Example 5.

FIG. 10 shows the fluorescence microscope observation results accordingto the cell test of the protein-coated polymer nanoparticles prepared inExample 5.

FIG. 11 is a schematic diagram showing the synthesis of the protein cageaccording to one embodiment of the present invention.

FIG. 12 shows TEM analysis results (top) and DLS data (bottom right)after a cross-linking agent (glutaraldehyde) is added to a solutioncomprising the polymer particles formed in Example 6.

FIG. 13 shows the TEM image and schematic view after the addition of THFto the cross-linked structure as confirmed in FIG. 12.

FIG. 14 shows the TEM image and schematic view after an excess ofimidazole is added when the inner polymer is melted out, as in FIG. 13,according to Example 7.

FIG. 15 shows the size and shape of the polymer-protein particlesprepared from Ni-NTA-PS and His6-GFP in a water-DMF solution (4% byvolume of DMF) according to Example 8. (A) is the DLS and TEM resultsafter removal of DMF by dialysis (24 h) and (B) is the DLS and TEMresults after the removal of DMF by dialysis (24 h) and the addition ofan excess imidazole solution (250 mM).

FIG. 16 shows the high resolution TEM images (A to C) and opticalfluorescence microscopy image (D) according to Example 8. (A) and (B)are images of the outer layers of polymer-protein particles, and (C) isan image of particles after controlling the inside polymers by theaddition of an imidazole solution of 250 mM. In (A), the protein layeroutside the particle can be clearly confirmed. The arrow in (B)indicates the outer polymer layer. The images are adapted from differentexperiments performed under the same experimental conditions.

FIG. 17 shows the change in the particle size depending on the polymerconcentration used in the preparation method of core-shell structuredpolymer-protein particles according to the present invention. Theexperiments were carried out at pH 8.0.

FIG. 18 shows the change in the particle size depending on the pH changein the preparation method of core-shell structured polymer-proteinparticles according to the present invention.

FIG. 19 shows the change of particle diameter versus the amount ofNi-NTA-PS (Mn of approx. 4,900) dissolved in 0.1 mL DMF, measured by DLSand TEM.

FIG. 20 shows the size and shape of the core-shell polystyrene-GFPparticles formed by the covalent bond between the NHS functional groupand the histidine tag as prepared in Example 10. The left side shows thesize distribution of particles measured by dynamic light scattering(DLS), and the right side shows the shape of particles observed by TEM.

FIG. 21 shows the size and shape of the core-shell polystyrene-RFPparticles formed by the covalent bond between the NHS functional groupand the histidine tag as prepared in Example 10. The left side shows thesize distribution of particles measured by dynamic light scattering(DLS), and the right side shows the shape of particles observed by TEM.

FIG. 22 shows the size and shape of the core-shell polystyrene-YFPparticles formed by the covalent bond between the NHS functional groupand the histidine tag as prepared in Example 10. The left side shows thesize distribution of the particles measured by dynamic light scattering(DLS), and the right side shows the shape of the particles observed byTEM.

FIG. 23 shows the size and shape of the core-shellpolystyrene-fibrinogen particles formed by the covalent bond between theNHS functional group and the histidine tag as prepared in Example 10.The left side shows the size distribution of the particles measured bydynamic light scattering (DLS), and the right side shows the shape ofthe particles observed by TEM.

FIG. 24 is a reaction scheme for the synthesis of polystyrene(tri-NTA-PS, 8′) wherein tri-NTA is bound to the terminal by ATRP (atomtransfer radical polymerization).

FIG. 25 shows ¹H NMR (A) and ¹³C NMR (B) spectra of a p-tri-NTAinitiator (6′).

FIG. 26 shows the analysis results by gel permeation chromatography (A)and MALDI-TOF mass spectrometry (B) of a p-tri-NTA initiator (6′).

FIG. 27 shows the ATRP results of styrene using a p-tri-NTA initiator(6′). Molecular weight (Mn) and dispersity (

) over time are shown together with the gel permeation chromatography oftri-NTA-PS (7′). The concentration ratio of the initial reactants was[Styrene]₀:[initiator]₀:[CuCl]₀:[dNbpy]₀=100:1:10:20, and anisole in 50%by volume was used as the solvent at 115° C.

FIG. 28 shows ¹H NMR (300 MHz) spectra of (A) p-tri-NTA-PS (Mn,GPC=6,400 g/mol,

=1.15; 7′) and (B) tri-NTA-PS (Mn, GPC=5,400 g/mol,

=1.17; 8′).

FIG. 29 shows ¹³C NMR (300 MHz) spectra of (A) p-tri-NTA-PS (Mn,GPC=6,400 g/mol,

=1.15; 7′) and (B) tri-NTA-PS (Mn, GPC=5,400 g/mol,

=1.17; 8′).

FIG. 30 shows the DLS data and TEM image of the spherical particleswhich are self-assembled from tri-NTA-PS in water/THF.

FIG. 31 shows the DLS data and TEM image of the polymer-proteincore-shell hybrid particles self-assembled from nickel-complexedtri-NTA-PS (Ni-tri-NTA-PS) and His6-GFP through the NTA-Ni/Hisinteraction in water/DMF (DMF 4 vol. %) according to Example 13.

BEST MODE FOR CARRYING OUT THE INVENTION

The first aspect of the present invention provides a method forpreparing a protein cage which comprises: a 1^(st) step of preparing anamphiphilic polymer comprising a 1^(st) hydrophobic polymer and a 1^(st)hydrophilic functional group; a 2^(nd) step of preparing a hydrophilicprotein comprising a 2^(nd) functional group binding to the 1^(st)functional group; a 3^(rd) step of forming an amphiphilicpolymer-protein hybrid by the binding of the 1^(st) functional group andthe 2^(nd) functional group, and forming core-shell structured particlescomprising a protein shell and an amphiphilic polymer core by theself-assembly of the amphiphilic polymer in a hydrophilic solvent; and a4^(th) step of removing some or all of the hydrophobic polymer of thecore part from the core-shell structured particles.

The second aspect of the present invention provides the protein cageprepared by the method of the first aspect.

The third aspect of the present invention provides an in situ method forpreparing hydrophobic additive-loaded core-shell structuredpolymer-protein particles which comprises: a 1^(st) step of preparing a1^(st) solution comprising an amphiphilic polymer comprising a 1^(st)hydrophobic polymer and one or more 1^(st) hydrophilic functional groupsand a hydrophobic additive in an organic solvent; a 2^(nd) step ofpreparing a 2^(nd) solution comprising a hydrophilic protein whichcarries a 2^(nd) functional group binding to the 1^(st) functional groupwhile maintaining its tertiary structure in a hydrophilic solventcomprising water, and a 3^(rd) step of mixing the 1^(st) solution intothe 2^(nd) solution, wherein, in a hydrophilic solvent, an amphiphilicpolymer-protein hybrid is formed through the binding of the 1^(st)functional group and the 2^(nd) functional group, and at the same timethe core-shell structured particles are formed by the self-assembly ofthe amphiphilic polymer to have the protein shell maintaining itstertiary structure and the core comprising the amphiphilic polymer andthe hydrophobic additive.

The fourth aspect of the present invention provides the hydrophobicadditive-loaded core-shell structured polymer-protein particles preparedby the method of the third aspect wherein the individual protein formingthe shell maintains its tertiary structure.

The fifth aspect of the present invention provides a drug deliverysystem which comprises the protein cage described in the second aspect;and a drug enclosed inside the cage, interposed between the proteins, orbound onto the surface of the cage.

The sixth aspect of the present invention provides a cosmeticcomposition which comprises the protein cage described in the secondaspect; and a cosmetic material enclosed inside the cage, interposedbetween the proteins, or bound onto the surface of the cage.

The seventh aspect of the present invention provides a composition forimaging which comprises the protein cage described in the second aspect;and a contrast agent enclosed inside the cage, interposed between theproteins, or bound onto the surface of the cage.

The eighth aspect of the present invention provides an artificialvaccine which comprises the protein cage described in the second aspect,wherein some or all of the proteins forming the protein cage areantigenic proteins.

The ninth aspect of the present invention provides a biosensor whichcomprises the protein cage described in the second aspect, wherein theprotein comprises two or more types of proteins.

Below, the present invention will be explained in more detail.

The “protein cage” as used herein forms an outer surface of a specificstructure through the gathering of two or more protein molecules, and itmay be used interchangeably with the protein shell.

In addition, the explanation of the “protein cage” may be applied to theprotein cage prepared in accordance with the first aspect of the presentinvention, as well as to the protein shell of the hydrophobicadditive-loaded core-shell structured polymer-protein particles preparedin the third aspect.

A protein has a unique amino acid sequence. This sequence is called aprimary structure and determines the structure and function of theprotein. Through the interaction of amino acids, protein chains form adistinctive secondary structure, and in some cases a tertiary structure.The secondary structure is determined by the angle of peptide bondslinking amino acids to each other, and this bond angle is made by thehydrogen bond between the nitrogen atom of one amino acid and the oxygenatom of another amino acid. In general, these hydrogen bonds form thehelical secondary structure. The tertiary structure is formed by foldingand bending of the protein chain to form a more or less sphericalprotein. The tertiary structure is determined by the side chains ofamino acids. There are side chains which are very bulky and thus destroythe normal secondary helical structure of the protein chain to causebending or twisting. Also, the side chains form an ionic bond byattracting each other when they have different charge, and repel eachother when they have the same charge. A water-insoluble hydrophobic sidechain tends to gather inside the protein and to avoid the outer partwhich is exposed to water. A hydrophilic side chain easily makes ahydrogen bond with a water molecule and is located in the outer part. Adisulfide bridge is a kind of covalent bond that is established betweentwo cysteines, which are an amino acid containing sulfur (—S—). Thedisulfide bridge thus formed (—S—) stabilizes the loop structure of theprotein chain.

The present invention utilizes the principle of manufacturing ahydrophobic polymer-hydrophilic protein hybrid core-shell structure byself-assembly in order to manufacture the protein cage artificially andto prepare the hydrophobic additive-loaded core-shell structuredpolymer-protein particles in situ in a one-pot reaction. The inventorshave found that some or all of the polymers can be removed from thehydrophobic polymer-hydrophilic protein hybrid core-shell structure,during which the proteins constituting the shell maintain the shape ofshell. They also have found that the polymer nanoparticles coated withprotein are formed by the one-pot encapsulation method and at the sametime the hydrophobic additive can be loaded by the hydrophobic polymer.Furthermore, they have found that, in a hydrophilic solvent (e.g., anenvironment appropriate for the physiological condition), the proteinmay be bound with the hydrophobic polymer while maintaining its tertiarystructure, and formed the protein shell of the core-shell structurethrough self-assembly, and the hydrophobic additive may be collected inthe core part simultaneously. The present invention is based on thisdiscovery.

The method for preparing a protein cage according to the first aspect ofthe present invention comprises: a 1^(st) step of preparing anamphiphilic polymer comprising a 1^(st) hydrophobic polymer and a 1^(st)hydrophilic functional group; a 2^(nd) step of preparing a hydrophilicprotein comprising a 2^(nd) functional group binding to the 1^(st)functional group; a 3^(rd) step of forming an amphiphilicpolymer-protein hybrid by the binding of the 1^(st) functional group andthe 2^(nd) functional group, and forming core-shell structured particlescomprising a protein shell and an amphiphilic polymer core by theself-assembly of the amphiphilic polymer in a hydrophilic solvent; and a4^(th) step of removing some or all of the hydrophobic polymer of thecore part from the core-shell structured particles.

In addition, the in situ method according to the third aspect of thepresent invention for preparing hydrophobic additive-loaded core-shellstructured polymer-protein particles comprises a 1^(st) step ofpreparing a 1^(st) solution comprising an amphiphilic polymer comprisinga 1^(st) hydrophobic polymer and one or more 1^(st) hydrophilicfunctional groups and a hydrophobic additive in an organic solvent; a2^(nd) step of preparing a 2^(nd) solution comprising a hydrophilicprotein which carries a 2^(nd) functional group binding to the 1^(st)functional group while maintaining its tertiary structure in ahydrophilic solvent comprising water, and a 3^(rd) step of mixing the1^(st) solution into the 2^(nd) solution.

The present invention is characterized in that the 1^(st) hydrophobicpolymer carries the 1^(st) hydrophilic functional group to form theamphiphilic polymer, the hydrophilic protein carries the 2^(nd)functional group binding to the 1^(st) functional group, and the 1^(st)hydrophobic polymer is connected with the hydrophilic protein throughthe binding of the 1^(st) and the 2^(nd) functional groups to form theamphiphilic polymer-protein hybrid (FIG. 1A). The 1^(st) functionalgroup must be hydrophilic to be able to guide the 1^(st) hydrophobicpolymer to the interface of the hydrophilic solvent where thehydrophilic protein is included and to be easily bound to the 2^(nd)functional group of the hydrophilic protein in the hydrophilic solvent.

The present invention is also characterized in that the amphiphilicpolymer-protein hybrid is formed through the binding of the 1^(st) andthe 2^(nd) functional groups in the hydrophilic solvent, and at the sametime the core-shell structured particles consisting of the protein shelland the core comprising the 1^(st) hydrophobic polymer are formedthrough the self-assembly of the amphiphilic polymer comprising the1^(st) hydrophobic polymer part which tends to be aggregated in thehydrophilic solvent (FIG. 1B). Here, since the protein can maintain itsown tertiary structure, it can still exhibit the activity of the proteinitself.

In order to synthesize a well-defined core-shell structure, theamphiphilic polymer comprising a 1^(st) hydrophobic polymer and a 1^(st)hydrophilic functional group may comprise one or more 1^(st) hydrophilicfunctional groups, but it is preferable that the hydrophilic proteincarries only one 2^(nd) functional group. This is because theorientation of hydrophilic protein in the protein cage or a proteinshell can be controlled as desired when one 2^(nd) functional group islimitedly connected to a specific part of the protein.

In particular, in order to maintain the unique activity of protein inthe protein cage or protein shell, it is better for the 2^(nd)functional group to be connected to a part that is not the proteinactive site, preferably to a three-dimensionally spaced part from theactive site in order for the site not being sterically hindered. Forexample, the 2^(nd) functional group can be connected to the N- orC-terminal if the active site is not in the terminal.

With the preparation method according to the present invention,different proteins as well as the same proteins may co-exist and/or beconcentrated in the protein shell part of the core-shell structure or ina certain space of a structure such as the protein cage. Thus, one typeof protein may make up the protein cage or protein shell, but two ormore types of proteins may be used in combination depending on thepurpose.

If the self-assembled polymer-protein hybrid nanostructure isestablished according to the present invention, some biofunctionalitiesmay be built into the nanostructure, and morphological architectures invarious shapes may be formed. In particular, since the size and shapemay be controlled during the formation of a polymer-protein hybridnanostructure, this nanostructure may be applied to the field of ananoreactor for a catalyst, as well as in the various biomedical sciencefields such as delivery of drugs, therapeutic agents, or diagnosticpreparations.

A series of experiments have been performed by changing variousparameters in order to control the size of polymer-protein hybridaggregates and to understand the mechanism of the in situ method ofpreparing the polymer-protein hybrid aggregates for potential biomedicalapplications. For example, similar experiments have been performed byusing His-tagged lipase instead of His6-GFP as a protein, usingNi-NTA-PS having a different molecular weight, changing theconcentration of the polymer and/or protein solution, adjusting the rateof addition of the polymer solution, using a different solvent, orremoving the organic solvent by dialysis.

As a result, the shape or size of the core-shell structured particlesmay be achieved by controlling the type/composition ratio, molecularweight, or concentration of the hydrophobic polymer; thetype/composition ratio, molecular weight, or concentration of theprotein; or the mixing ratio or mixing rate of the hydrophobic polymerand protein (Table 1). For example, as the ratio of the polymer and theprotein changes, the rate of bond formation between the polymer and theprotein and the rate of particle formation by self-assembly change,thereby determining the size of the finally generated core-shellstructured polymer-protein particles. Here, the proteins have one 2^(nd)functional group which binds to the 1^(st) functional group for eachmolecule and are competitively bound. Therefore, when two or moreproteins are used, it is possible to control the composition ratio ofthe protein that makes up the protein shell by adjusting mixing ratiothereof.

The core-shell structured polymer-protein particles formed according tothe preparation method of the present invention may have an averagediameter of 20 nm to 5 μm. In addition, the particles may be prepared ina spherical, oval, or rod shape, but the shape is not limited thereto.

In a case where a protein cage is prepared according to the presentinvention, it may be synthesized with a well-defined structure by asimple process, and few constraints on the selection and introduction ofproteins make the cage available in various ways.

Furthermore, during the preparation of protein cage according to thepresent invention, the formation of particles coated by a protein andthe encapsulation (loading) of the hydrophobic additive may be performedat the same time by a one-pot reaction. Thus, the process is very simpleand can be effectively used in a variety of applications includingdelivery systems of drugs, cosmetic materials, etc. Also, fewconstraints on the selection of the substance to be loaded in theprotein cage and the type of polymer used make it possible to select thesubstance from a wide range.

The 1^(st) functional group, the 2^(nd) functional group, or both may beconnected directly or via a linker to the polymer and the protein,respectively.

Non-limiting examples of the binding between the 1^(st) functional groupand the 2^(nd) functional group include a coordinate bond, a covalentbond, a metallic bond, a hydrogen bond, an ionic bond, anantigen-antibody binding, a ligand-receptor binding, etc. (FIG. 1C). Thebinding between the 1^(st) functional group and the 2^(nd) functionalgroup is preferably specific.

A polymer having IMAL (immobilized-metal affinity ligand) at itsterminal may be exemplified as the 1^(st) hydrophobic polymer having the1^(st) functional group, and a protein to which the above IMAL-affinitytag is attached may be exemplified as the hydrophilic protein having the2^(nd) functional group binding to the 1^(st) functional group. IMAL isa ligand containing a transition metal such as Ni²⁺, Co²⁺, Zn²⁺, etc.,preferably Ni²⁺. As preferable examples thereof, Ni-NTA(nitrilotriacetic acid), Ni-IDA (iminodiacetic acid), Ni-TED(tris(carboxymethyl)ethylene diamine), etc. may be mentioned. Also,preferably, the IMAL-affinity tag may be a histidine tag having animidazole ring, which is affinitive to the metal ion, at the side chain.The polymer-protein hybrid may be formed through a coordinate bondbetween the histidine tag and Ni-NTA (FIG. 4B).

Or, conversely, a polymer combined with histidine as the IMAL-affinitytag and a protein combined with IMAL may be used.

Other specific examples of the 1^(st) and the 2^(nd) functional groupsthat can be bound to each other include the covalent bond with NHS(N-hydroxysuccinimidyl 2-bromo-2-methyl propionate) and theligand-receptor binding between biotin and avidin, etc. An NHSfunctional group may form a covalent bond with a primary amine. Thus, apolymer containing NHS may form a covalent bond via such amino acidresidues as arginine, lysine, asparagine, glutamine, etc., each of whichcontains a primary amine in the side chain. Or, the protein modified tohave the NHS functional group may form a covalent bond with a polymercontaining a primary amine group. Meanwhile, the ligand-receptor bindingbetween biotin and avidin may be achieved between a polymer modifiedwith biotin and a protein containing avidin, or between a polymer and aprotein conversely modified. Alternatively, since the avidin has aplurality of binding sites to biotin, it may be a form wherein thebiotinylated polymer is bound to the protein through the avidin. Theavidin includes avidin, streptavidin, and deglycosylated avidin(NeutrAvidin) without limitation.

In order to form the core-shell structured particle such as a micelle ina hydrophilic solvent through self-assembly, there is no limitation ontypes of the 1^(st) polymer and the protein in the present inventiononly if the 1^(st) polymer is hydrophobic enough to be aggregated in ahydrophilic solvent, the protein is hydrophilic enough to be dispersedor dissolved uniformly in the hydrophilic solvent, and the 1^(st)polymer and the protein have the 1^(st) functional group and the 2^(nd)functional group, respectively, that can be bound to each other.

The 1^(st) polymer is preferably a biocompatible and/or biodegradablepolymer, and may be selected from polyglycolide (PGA), polylactide(PLA), polymethylmethacrylate (PMMA), polystyrene, poly(meth)acrylate(PMA), polycaprolactone (PCL), and derivatives thereof. Meanwhile,non-limiting examples of the monomer which forms the polymer includestyrene, acrylate, lactide, hydroxybutyric acid, etc.

In the present invention, the protein includes a conjugated proteincomprising a non-amino acid prosthetic group, as well as a simpleprotein consisting only of amino acids. As the prosthetic group,carbohydrates, lipids, nucleic acids, metals, pigments, etc. and somenon-protein molecules, ions, etc. can be mentioned. Also, in the presentinvention, the protein includes structural proteins (e.g., collagen,keratin, etc.), biologically active proteins (enzymes, hormones,transport proteins of materials, immunoglobulins, etc.), and fragmentsof proteins (e.g., a variety of motifs such as enzyme active sites,binding sites, functional sites, etc.).

In addition, proteins, peptides, motifs, fusion proteins, peptidederivatives, proteins modified by PEG, etc., synthetic proteins, andnatural proteins also belong to the category of the protein in thepresent invention. Even if the protein has a hydrophobic moiety withinit, as long as the protein surface is hydrophilic and the protein can beuniformly dispersed in a hydrophilic solvent, it belongs to the categoryof the hydrophilic protein in the present invention. In particular, itis preferable that the hydrophilic protein has a hydrophilic propertywhile maintaining its tertiary structure or the three-dimensionalconformation in the hydrophilic solvent.

Non-limiting examples of the protein include human growth hormone, G-CSF(granulocyte colony stimulating factor), GM-CSF (granulocyte-macrophagecolony-stimulating factor), erythropoietin, vaccines, antibodies,insulin, glucagon, calcitonin, ACTH (adrenocorticotropic hormone),somatostatin, somatotropin, somatomedin, parathyroid hormone, thyroidhormone, hypothalamus secrete substances, prolactin, endorphin, VEGF(vascular endothelial growth factor), enkephalin, vasopressin, nervegrowth factor, non-naturally occurring opioid, interferon, asparaginase,alginase, superoxide dismutase, trypsin, chymotrypsin, pepsin, etc.

The hydrophilic solvent is not limited as long as the amphiphilicpolymer-protein hybrid can form a core-shell structure throughself-assembly. Non-limiting examples of the hydrophilic solvent includewater or a solvent mixture thereof. However, it is preferable to use asolvent in which the tertiary structure of the protein, i.e., theactivity of the protein can be maintained, more preferable to use asolvent having the pH and/or temperature range corresponding to thephysiological conditions, and still more preferable to use a pH buffer(e.g., phosphate buffer solution) so that the shell-forming protein mayform the protein shell under the circumstance where the protein canexert its own function.

The organic solvent is not limited as long as it can dissolve ordisperse the amphiphilic polymer comprising the 1^(st) hydrophobicpolymer and the 1^(st) functional group therein. Non-limiting examplesof the organic solvent include a C1 to C6 alcohol, acetone, DMF(dimethylformamide), DMSO (dimethyl sulfoxide), THF (tetrahydrofuran),etc.

The organic solvent may be trapped in the core with the hydrophobicpolymer during the formation of the core-shell particles. Thus, if ahydrophobic additive is dissolved or dispersed in the organic solvent,it may be trapped in the core with the organic solvent.

Based on the confirmation that the formation of (hydrophobic) polymernanoparticles coated with a protein and the loading of a hydrophobicadditive in the hydrophobic polymer can be simultaneously performed by aone-pot encapsulating method in a single step of mixing the 1^(st) andthe 2^(nd) solutions, the third aspect of the present invention ischaracterized in that the hydrophobic additive to be loaded in the corepart is further added to the 1^(st) solution having the amphiphilicpolymer comprising the 1^(st) hydrophobic polymer and the 1^(st)hydrophilic functional group in an organic solvent in the in situ methodfor preparing the core-shell structured polymer-protein particles.

In addition, since the preparation method according to the third aspectof the present invention can manufacture the polymer-protein hybridnanostructure by self-assembly in a hydrophilic solvent in an in situone-pot reaction, the type, molecular weight, or concentration of thehydrophobic polymer; the type, molecular weight, or concentration of theprotein; or the mixing ratio or mixing rate of the hydrophobic polymerand the protein may be controlled to adjust the shape and/or size of theparticles formed. Thus, it is also another feature that the shape and/orsize of the hydrophobic additive-loaded core-shell structuredpolymer-protein particles prepared by the in situ method according tothe present invention can be adjusted.

Although the additive itself is hydrophilic, if the surface thereof ismodified to be hydrophobic, it can be used as a hydrophobic additive.

The hydrophobic additive can be a drug, and thus particles loaded withthe drug as a hydrophobic additive may be used as a drug deliverysystem. The earlier hydrophobic drugs are sparingly soluble and thus canhardly be administered. The present invention can address this problemby making the hydrophobic drug to be loaded in a particle having asurface coated with the hydrophilic protein. In particular, since thepreparation method of the present invention proceeds in a single-stepreaction wherein the hydrophobic drug is dissolved together with thepolymer in an organic solvent and then mixed with a solution wherein theprotein is dissolved in a hydrophilic solvent, it can provide a drugdelivery system in the form of hydrophobic additive-loaded core-shellstructured polymer-protein particles in a fast and convenient manner.

Non-limiting examples of the drug include anti-cancer agents such aspaclitaxel, methotrexate, doxorubicin, 5-fluorouracil, mitomycin-C,styrene maleic acid neocarzinostatin, cisplatin, carboplatin,carmustine, dicarbazine, etoposide, daunomycin, etc.; anti-viral agents;steroidal anti-inflammatory drugs; antibiotics; antifungal agents;vitamins; prostacyclins; antimetabolic agents; cholinergic agents;adrenergic antagonists; anti-convulsants; anxiolytics; tranquilizers;antidepressants; anesthetics; pain relievers; anabolic steroids;immunosuppressive agents, immune enhancers, etc.

The hydrophobic additive may be a cosmetic material. The “cosmeticmaterial” is defined as a material which is used for the human body toclean and beautify the body for the purpose of adding attractiveness,brightening the appearance, or maintaining or promoting the health ofskin and hair, and has little action on the human body. Non-limitingexamples of the cosmetic material include emollients, preservatives,fragrance substances, anti-acne agents, antifungal agents, antioxidants,deodorants, anhidrotic agents, anti-dandruff agents, decolorants,antiseborrheic agents, dyes, suntan lotions, UV light absorbers,enzymes, aroma substances, etc.

Likewise, the hydrophobic additive may be a contrast agent. The“contrast agent” is a substance that functions to provide a clear imagefor the sites that cannot be confirmed by simple imaging, therebyallowing early diagnosis and treatment of the disease occurring at thosesites. Contrast agents for magnetic resonance imaging (MRI), computedtomography (CT), positron emission tomography (PET), ultrasonography,fluoroscopy, etc. may be used. Non-limiting examples of the contrastagent include those for magnetic resonance imaging (MRI) which areparamagnetic or superparamagnetic materials of transition metal ionssuch as gadolinium (Gd), manganese (Mn), copper (Cu), and chromium (Cr),hydrophobic complexes of the transition metal ions such as gadopentetatedimeglumine (Gd-DTPA) and gadoterate meglumine (Gd-DOTA),fluorine-containing compounds such as perfluorocarbons andperfluoropropane, iron oxide-, manganese-, copper-, and chromium-basednanoparticles, and nanoparticles whose surface is modified byhydrophobic substances; those for computed tomography (CT) which areiodinated hydrophobic material derived from iodinated poppy seed oil,nanoparticles consisting of a metal element comprising bismuth (Bi),gold (Au), silver (Ag), etc.; those for positron emission tomography(PET) which are radioactive isotopes comprising ^(99m)Tc, ¹²³I, ¹⁶⁶Ho,¹¹¹In, ⁹⁰Y, ¹⁵³Sm, ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁸Ga, and ¹⁷⁷Lu, and hydrophobiccomplexes of the radioactive isotopes prepared by using diethylenetriamine pentaacetate (DTPA); those for ultrasonography which arehydrophobic compounds such as perfluoropropane, perfluorohexane, sulfurhexafluoride, perfluoropentane, decafluorobutane, etc.; and those forfluoroscopy which are fluorescein, rhodamine, Nile red, Cy-3, Cy-5, etc.

Ni-NTA is an example of the 1^(st) functional group in the 1^(st)hydrophobic polymer carrying the 1^(st) functional group. The synthesismechanism of the polymer wherein Ni-NTA is bound to its terminal isschematically illustrated in FIG. 4A (Preparation Example 1). Meanwhile,FIG. 4C shows a schematic diagram of a process wherein the amphiphilicpolymer-protein hybrid is formed through the binding of the 1^(st) andthe 2^(nd) functional groups, and then the hydrophobic additive-loadedcore-shell structured particles are formed by self-assembly of thehybrid in a hydrophilic solvent.

Referring to FIGS. 4A and 4C, an initiator of the R—X type having NTA issynthesized, a Ni-NTA-polymer is synthesized through ATRP livingpolymerization, and then a solution of the Ni-NTA-polymer and ahydrophobic material (Nile red) in DMF is added dropwise to a PBS buffersolution wherein the protein with a histidine tag is dissolved to formprotein-coated polymer particles and concurrently to provide thehydrophobic material-loaded core. This action is caused by theinteraction between the histidine tag and Ni-NTA as well as theaggregation of the polymer in water.

In an example of the present invention, the Nile red dye was used as ahydrophobic additive to confirm whether it is enclosed in the proteincage and the possibility of delivery into the inside of a cell (FIGS. 9and 10).

In the present invention, a 2^(nd) hydrophobic polymer not having the1^(st) functional group may be further added during the preparation ofthe core-shell structured polymer-protein particles. For this purpose,the 1^(st) solution used for the preparation of the core-shellstructured polymer-protein particles according to the present inventionmay further comprise the 2^(nd) polymer not having the 1^(st) functionalgroup.

The 2^(nd) hydrophobic polymer may be located together with the 1^(st)hydrophobic polymer in the core part of the core-shell structuredparticles to control the size of particles, and further it is not boundor less bound than the 1^(st) hydrophobic polymer to the amphiphilicpolymer-protein hybrid, and can thereby be easily removed from thecore-shell structured particles later. The 2^(nd) hydrophobic polymermay be the same as or different from the 1^(st) hydrophobic polymer. Inshort, the 2^(nd) hydrophobic polymer not having the 1^(st) functionalgroup binding to the protein but having only the hydrophobic moiety ofthe 1^(st) polymer may be further added in preparing the core-shellstructured polymer-protein particles in order to control the particlesize and/or the number of proteins bound per single particle.

In addition, the preparation method according to the present inventionmay further comprise a step of forming bindings between shell-formingproteins by adding a cross-linking agent to the core-shell structuredparticles which are formed in the 3^(rd) step.

If a cross-linking agent is further added, cross-linking occurs betweenthe proteins to further stabilize the core-shell particles and to makethe encapsulation by the protein shell easier. As the cross-linkingagent, glutaraldehyde, NHS ester, EDC, maleimide, pyridyl disulfide,hydrazide, alkoxy amines, etc. may be used.

The fourth aspect of the present invention is characterized in that itfurther comprises the 4^(th) step of removing some or all of thehydrophobic polymer of the core part from the core-shell structuredparticles in order to generate a protein cage.

Removal of some or all of the hydrophobic polymer of the core part fromthe core-shell structured particles may be performed by the introductionof (i) a competitor compound for the binding between the 1^(st) and the2^(nd) functional groups, or (ii) a compound that hydrolyzes the polymermoiety in the amphiphilic polymer-protein hybrid.

FIG. 11 shows a schematic diagram for the preparation of the proteincage according to one embodiment of the present invention.Protein-coated polymer particles are formed when a solution of thepolymer with Ni-NTA at its terminal in DMF is added dropwise to a PBSbuffer solution wherein the protein with a histidine tag is dissolved.The protein in the surface of the particles thus formed is cross-linked,and the particles are dissolved in an organic solvent to remove theinner polymer dissolved away to the outside of the particle. Here, adifference is observed depending on whether the binding between Ni-NTAand the histidine tag is dissociated or not. If an excess of imidazoleis added to the solution and reacted, the excess imidazole is bound toIMAL competitively with the protein having an IMAL-affinity tag toreplace the binding of polymer and protein, thereby eluting only thepolymer from the core-shell particles.

As described above, in a case where the polymer and protein areconnected by non-covalent binding in the amphiphilic polymer-proteinhybrid, the binding may be easily dissociated by adding an excess of amaterial which can competitively replace the binding. Therefore,preferably, after the protein shell is cross-linked, the inner polymeris separated and removed by using the above competitive reagent tofinally synthesize a protein cage.

Meanwhile, in a case where the polymer and protein are connected by acovalent bond in the amphiphilic polymer-protein hybrid, the polymeroccupying the particle core may be removed by using a reagent, asolvent, or a solution containing it, each of which is capable ofdecomposing the polymer.

The protein cage which can be prepared according to the presentinvention may preferably have a diameter of 20 nm to 5 μm, but is notlimited thereto. Also, the protein cage according to the presentinvention may have a spherical, oval, or rod shape.

The protein cage/protein shell prepared by the method of the first orthe third aspect of the present invention can play a role as ananostructure of a functional protein having a specific bindingcapacity, catalytic capacity, etc. For example, protein nanostructureshaving various functions may be provided depending on the selection of astructural protein of the cage (FIG. 2), and furthermore proteinnanostructures that can be utilized in various applications such as adelivery system for an active component (physiologically activecomponent, drug, etc.), a sensor, a catalyst, etc. may be provideddepending on the function of the additive loaded (FIG. 3).

The protein cage according to the present invention can encapsulate ahydrophobic additive such as a sparingly soluble drug. The hydrophobicadditive may be added in the 3^(rd) step to be loaded in the core partduring the self-assembly, or may be injected into the protein cageformed in the 4^(th) step. When the additive is injected into theprotein cage which is prepared according to the first aspect, it is notlimited to the hydrophobic one, but a hydrophilic additive can be used.

Since the cage-forming protein can be a single protein or amulti-protein, it is possible to provide a cage having a monofunctionalor multifunctional protein.

Non-limiting examples of the cage-forming protein include asensor/reporter protein (sensor protein; e.g., green fluorescentprotein), an enzyme (e.g., lipase, esterase, horseradish peroxidase), atarget-oriented body protein (recognition protein), a vaccine protein(e.g., antigen, hemagglutinin), a skin-functional/permeable peptide, andderivatives thereof.

Thus, the protein cage according to the present invention can provide avaccine superior to the virus-like particle (VLP) (fast responsiveness,reduced side effects), a biological target cell-oriented contrast agent,a protein carrier for a functional substance (drugs, functionalsubstances for skin, healthcare compounds), or a target-oriented proteincarrier for a functional protein (in the case of a multi-protein cage)by appropriately selecting the cage-forming structural protein.

The protein cage prepared by the method of the first aspect or the thirdaspect may be used for a drug delivery system which comprises a drugenclosed inside the cage, interposed between the proteins, or bound ontothe surface of the cage; a cosmetic composition which comprises acosmetic material enclosed inside the cage, interposed between theproteins, or bound onto the surface of the cage; or a composition forimaging which comprises a contrast agent enclosed inside the cage,interposed between the proteins, or bound onto the surface of the cage.

The drug is conventionally a substance showing a specific prevention ortreatment effect against a specific disease, and it may show toxicityfor normal cells other than the target tissue in some cases. Thus,delivering these drugs specifically to a site in need thereof is animportant factor in minimizing side effects of the drug and maximizing aprevention or treatment effect thereof. Therefore, the protein making upthe protein cage/protein shell may itself be a targetable protein, ormay be a protein to which another antigen, antibody, ligand, or receptorbeing targetable is bound. In this case, targeting the drug containedtherein to the desired site is possible. Also, when used as acomposition for imaging, it is desirable that the protein making up theprotein cage/protein shell is itself a protein being targetable or aprotein to which another antigen, antibody, ligand, or receptor beingtargetable is bound, in order for it to be specifically moved to thetissue to be imaged, like a drug delivery system.

Meanwhile, it is preferable that the cosmetic material can be deliveredinside the skin by passing through the epidermal layer in order to showthe effect when applied to the skin. Thus, to effectively deliver thecosmetic material inside the skin, the protein making up the proteincage/protein shell is itself a protein having skin permeability or is aprotein to which a skin-permeable peptide or compound is bound.

Skin-permeable peptides that can be used to promote skin permeation areexemplified in U.S. Pat. No. 7,659,252 (incorporated in the presentspecification). These peptides exhibit an excellent skin permeationrate, and further can be used as a carrier for the transdermal deliveryof other drugs.

The protein cage prepared by the method of the first aspect or the thirdaspect can be used as an artificial vaccine when some or all of theproteins are an antigenic protein. Since the protein cage according tothe present invention is much larger in size than the individualprotein, it has an excellent immunity as a vaccine. In addition, thevaccine production time can be reduced, rapid development of the vaccineis possible, the particle size can be adjusted, and the potential tocause immune side effects may be reduced.

An example of the artificial vaccine that can be prepared according tothe present invention is schematically depicted in FIG. 3. For example,if the self-assembled protein cage according to the present invention isprepared using the HA/NA antigenic protein as an influenza surfaceprotein, it is possible to provide a highly efficient influenzapreventive vaccine.

Also, the protein cage prepared by the method of the first aspect or thethird aspect may include such proteins as enzymes, antigens, antibodies,ligands, or receptors which cause some physico-chemical changes in thesubstance to be detected or undergo some physico-chemical changes by thesubstance to be detected, and thus may be used as a biosensor. Ifappropriate, two or more types of proteins may be used as the protein.For example, the protein cage according to the present invention mayprovide a sensor in the form that the proteins for detection such asenzymes are fixed on the spherical support. Furthermore, a coenzyme or amaterial to be further required in a reaction may be loaded inside ofthe cage, thereby representing a relatively high concentration thereofin the cage and thus showing the effect of enhancing the local signal.

The glucose enzyme sensor is an example of a biosensor, and is based onthe phenomenon that glucose is converted to glutamic acid by the glucoseoxidase with consuming oxygen and generating hydrogen peroxide.Therefore, the biosensor works in the manner of measuring the increasein the amount of charge, the pH change, the reduced amount of oxygen,etc. generated by the secondary oxidation of hydrogen peroxide. Suchreactions of consuming oxygen and generating hydrogen peroxide as aby-product are typical ones by a variety of oxidases (e.g., galactoseoxidase, lactate oxidase, cholesterol oxidase, etc.) besides the glucoseoxidase. Thus, the protein cage according to the present invention maybe prepared to be used as a sensor, that is, it may be a component of aproduct utilized as the existing biosensor in such a manner that anenzyme is suspended in a solution or supported/fixed on a membrane or asupport. As described above, since the protein cage prepared in this waycan fix tens to thousands or more of enzymes in a particle, it may showthe effect of enhancing the local signal and effectively detect even asmall amount of sample.

An illustrative example of a sensor utilizing a protein cage containinga heterologous protein may be the case wherein a peroxidase usinghydrogen peroxide generated from the oxidase reaction is furthercontained in addition to the oxidase. Since the peroxidase converts thesubstrate compound to a colored product, it is possible to determine theactivity of the oxidase by measuring the color change. In this case, thesubstrate compound of peroxidase may be enclosed inside the protein cagefor efficient detection.

Preferably, the biosensor may further have a detector. The detector maybe an electrochemical signal detector, an optical detector, a pHdetector, a gas detector, etc., each of which is known in the art,without limitation.

MODE FOR INVENTION

Hereinafter, the present invention will be explained in more detail bythe following examples. These examples are intended to illustrate thepresent invention more specifically, but the scope of the presentinvention is not limited thereto.

PREPARATION EXAMPLE 1 Preparation of Polymer with NI-NTA at its Terminal

A polymer having Ni-NTA at its terminal was synthesized as depicted inFIG. 4(A).

1.1. Synthesis of NTA Initiator (2)

2-Bromoisobutyryl bromide (0.09 mL, 0.85 mmol) was slowly introduced toa flask charged with Compound 1(N-(5-amino-1-carboxypentyl)iminodiacetic acid tri-t-butyl ester, 342mg, 0.77 mmol), triethylamine (0.32 mL, 2.3 mmol), and THF (50 mL) at 0°C. for 1 h. After introduction of the acid bromide was completed, thereaction mixture was reacted at room temperature for 12 h, THF wasremoved, and the reaction mixture was dissolved in 100 mL of methylenechloride and washed with distilled water (5×100 mL). The product waspurified by column chromatography (hexane:ethyl acetate=4:1). Thestructure of the product was analyzed by ¹H NMR.

¹H NMR (300 MHz, CDCl₃): δ 1.38 (s, 18H), 1.40 (s, 9H), 1.49 (m, 2H),1.60 (m, 2H), 1.65 (m, 2H), 1.88 (s, 6H) 3.20 (t, 2H), 3.25 (t, 1H),3.42 (dd, 4H).

1.2. Preparation of NTA-Functionalized Polystyrene (3)

Styrene (1.00 mL, 8.82 mmol, 104 g/mol) and anisole (1.00 mL) wereintroduced to a Schlenk flask charged with nitrogen, three cycles of afreeze-pump-thaw process were repeated, CuCl (36.4 mg, 0.368 mmol) anddNbpy (300.8 mg, 0.736 mmol) were added to the flask, and then twocycles of the freeze-pump-thaw process were further performed. The flaskwas placed in an oil bath at 110° C., and then the NTA initiator 2(106.5 mg, 0.184 mmol) was added to the reaction mixture. After thepolymerization was completed, Compound 3 was obtained by precipitationfrom methanol (Mn=6500 g/mol).

1.3. Removal of Protecting Group of (3) (4)

Compound 3 (300 mg, 0.06 mmol) and trifluoroacetic acid (0.14 mL, 1.86mmol) were dissolved in 20 mL of methylene chloride and then reacted atroom temperature for 12 h. After the solvent was removed, Compound 4 wasobtained by precipitation from methanol.

1.4. Formation of Complex with Nickel (5)

Compound 4 (100 mg, 0.02 mmol) was dissolved in 50 mL of DMF, nickelchloride (54.4 mg, 0.42 mmol) was added thereto, and the reaction forforming a complex with nickel was performed at room temperature for 12h. The reaction mixture was precipitated from methanol to give Product5.

EXAMPLE 1 Preparation of Protein-Coated Polymer Nanoparticles (6) byCoordinate Bond and Determination of Size of Protein-Coated PolymerNanoparticles

The product (5) (0.1 mg, 1.5×10⁻⁵ mmol) prepared in Preparation Example1 was dissolved in 0.2 mL of DMF, and the solution was added dropwise to5 mL of a phosphate buffer solution (10 mM, pH 7.5) containing His6-GFP(0.41 mg, 1.4×10⁻⁵ mmol), using a syringe pump at a rate of 0.02 mL/h atroom temperature. After addition over 10 h, the reaction mixture wasstirred for 1 day.

Preparation of TEM Sample

Carbon-coated copper grid was soaked in the solution comprising theprotein-coated polymer nanoparticles prepared in Example 1 to preparethe TEM sample. The excess solution was removed with the filter paper,and the grid was dried at room temperature for 6 h. The sample was notstained.

Preparation and Measurement of DLS Sample

A DLS test was performed for the protein-coated polymer nanoparticlesprepared in Example 1 utilizing a laser operating at 660 nm and anoptimized self-constructed setup. DLS samples were prepared by dilutingeach sample 10- or 20-fold with water-DMF (DMF 4 vol. %, pH 7.4). Theabove samples were loaded in spherical glass cuvettes beforemeasurement. All measurements were performed at 25° C. at 900. Eachmeasurement was the sum of 5 repetitions, and the single measurementtime was 1 min. The size of acquired hybrid particles was presented as anumber distribution.

FIG. 6 shows the TEM analysis results (A, B) and DLS data (C) to verifythe formation of the protein-coated polymer nanoparticles according toExample 1. As shown in TEM photograph (A), nanoparticles of uniformshape were formed, and upon magnifying (B), it could be found thatparticles were formed with a uniform shape inside and outside of theparticles, and the contrasts of the inside and outside of the particleswere different. In addition, DLS data (C) showed that there wereparticles of uniform size with a narrow distribution.

Meanwhile, the stability of the polymer-protein hybrid aggregates is animportant factor in potential applications thereof in the biologicalfield. In order to study the stability of the hybrid aggregates acquiredfrom Ni-NTA-PS (Mn of approx. 21,800) and His6-GFP, the solutioncontaining the polymer-protein hybrid colloid was continuously stirred,and DLS measurements were performed at uniform time intervals up to 1month. DLS and TEM studies showed that the aggregates appeared to bestable up to 15 days and degraded thereafter without phase separation(precipitation).

EXAMPLE 2

FIG. 5 shows the TEM analysis results (top) and DLS data (bottom), whichconfirmed that polystyrene particles bound with Ni-NTA were formeduniformly when polystyrene bound with Ni-NTA at its terminal (0.1 mg,1.5×10⁻⁵ mmol) and Nile red (0.02 mg, 6.3×10⁻⁵ mmol) dissolved in 0.2 mLof DMF were slowly added dropwise to 5 mL of a PBS buffer solutioncontaining His6-GFP (0.41 mg, 1.4×10⁻⁵ mmol), a protein having ahistidine tag. As shown in the TEM photograph, it can be found thatpolystyrene particles were formed in a uniform size and stabilized asthe proteins were bound to these particles. In addition, DLS data showedthat there were particles of uniform size.

EXAMPLE 3

Size of the resulting aggregates was determined, which changes accordingto the amount of the polymer dissolved in an organic solvent, unlikeExample 1.

0.25 mg of Ni-NTA-PS (Mn of approx. 21,800, 1.2×10⁻⁵ mmol) was dissolvedin 0.2 mL of DMF, and the above polymer solution was slowly added to 5mL of deionized water containing His6-GFP (27 kDa, 261 mg, 9.8×10⁻⁶mmol) at a rate of 0.02 mL/h to prepare the aggregates. The shape andsize of the prepared aggregates were measured and presented in FIG. 7.Under the reaction conditions described above, spherical aggregateshaving a size of 80 nm to 140 nm were formed, and the DLS data and therepresentative TEM image were shown in FIGS. 7A and 7B.

FIG. 7C is digital photographs showing the aqueous His6-GFP solution (pH7.4) before the addition of Ni-NTA-PS dissolved in DMF (1) and thecolloidal solution of polymer-protein particles prepared by the additionof Ni-NTA-PS dissolved in DMF (2). The polymer-protein hybrid colloidalsolution became less transparent compared to the His6-GFP solution dueto the formation of aggregates (FIG. 7C).

When the experiment was performed without His6-GFP, although thereaction was carried out under the same experimental condition,Ni-NTA-PS itself formed ill-defined large amorphous aggregates, whereaswhen the DMF solution of polystyrene modified by the nickel-complexedNTA at its terminal (Ni-NTA-PS) was slowly added to the aqueous solution(pH 7.4) containing His6-GFP using a syringe pump, spherical aggregateswere found to be produced due to the specific interactions between theprotein and the polymer.

EXAMPLE 4 Preparation of Enzyme-Coated Polymer Nanoparticles

Enzyme-coated polymer nanoparticles were prepared by a method similar tothat described in Example 1.

Specifically, polystyrene modified by the nickel-complexed NTA at itsterminal (Ni-NTA-PS, Mn of approx. 21,800, 0.0625 mg, 3.0×10⁻⁶ mmol) wasprepared by stepwise dilution in 0.05 mL of DMF. In a glass vial underagitation, the polymer solution was slowly added to 1.25 mL of deionizedwater containing His-tagged enzyme (His6-Lip21H, 37 kDa, 77.5 mg,2.1×10⁶ mmol) at a rate of 0.02 mL/h using a syringe pump at roomtemperature (22° C.). Several drops of phosphate buffer saline (PBS, 50mM, pH of approx. 7.4) were added to maintain the pH at 7.4 before thepolymer solution was added to a water-soluble enzyme solution. After theaddition of the polymer solution was completed, the resultingpolymer-enzyme hybrid solution was continuously stirred, and theself-assembled form was analyzed by DLS and TEM. Next, a similar processwas performed for the conjugation with His6-Lip83H (27 kDa).

DLS and TEM analysis results for the above prepared enzyme-coatedpolymer nanoparticles were presented in FIGS. 8. (A) and (B) are theresults for the particles prepared utilizing His6-Lip21H (37 kDa) andHis6-Lip83H (27 kDa) as the histidine-tagged enzyme, respectively. Asshown in FIG. 8, His6-Lip21H (FIG. 2A; 37 kDa)-coated particlesexhibited the size distribution in the range of 90 nm to 150 nm, andHis6-Lip83H (FIG. 2B; 27 kDa)-coated particles exhibited the sizedistribution in the range of 70 nm to 120 nm, which was somewhat lowerthan the former.

EXAMPLE 5 Hydrophobic Dye-Loaded Protein-Coated Polymer Nanoparticles(6)

The product 5 (0.1 mg, 1.5×10⁻⁵ mmol) prepared in Preparation Example 1and Nile red (0.02 mg, 6.3×10⁻⁵ mmol) were dissolved in 0.2 mL of DMF,and this solution was added to 5 mL of a phosphate buffer solutioncontaining His6-GFP (0.41 mg, 1.4×10⁻⁵ mmol), using a syringe pump at arate of 0.02 mL/h at room temperature. After addition over 10 h, thereaction mixture was stirred for 1 day, and the Nile red which had notbeen encapsulated was removed using a 200 nm membrane syringe filter.

FIG. 9 shows the results of emission spectrum measurements, from whichit was confirmed that Nile red has the characteristic of its wavelengthrange being shifted to a short-wavelength region when it is loaded (FIG.9b ). If unloaded Nile red was removed through filtration, the peaks ofa long-wavelength region disappeared and GFP appeared to retain itsfluorescence during the process (FIG. 9a ). Also, Nile red was confirmedby fluorescence microscopy to remain even after the filtration.

In order to investigate whether the particles prepared in Example 5 canenter the inside of the cell, observation by fluorescence microscopy wasconducted. As depicted in FIG. 10, the presence of both GFP and Nile redwas confirmed, which exhibit intracellular green fluorescence and redfluorescence, respectively.

EXAMPLE 6 Cross-Linking of Protein Coating Shell of Protein-CoatedPolymer Nanoparticles (8)

0.1 mL of a 2.5% glutaraldehyde aqueous solution was injected into thesolution prepared according to Example 1 using a syringe pump for 30 minat room temperature. After 30 min, the reaction was stopped using sodiumborohydride.

FIG. 12 shows the TEM analysis results (top) and DLS data (bottom right)after the addition of a cross-linking agent (glutaraldehyde) to thesolution in which the core-shell structured polymer-protein particleshad formed. As shown in the TEM photograph, cross-linking of proteinsoccurred, the particles thereby became more stable, and subsequentpurification through a centrifugal filter removed the reactants, bufferions, etc., giving a clean solution where only particles were present inthe water phase.

EXAMPLE 7 Preparation of Protein Nanocage (9) (10)

FIG. 13 shows the TEM photograph and schematic diagram (9) after theaddition of THF to the cross-linked structure confirmed in FIG. 12.Here, the polystyrene which had aggregated inside the cross-linkedstructure was eluted out by the addition of THF, but at this time, thepolymer bound to the protein still remained inside the structure.

Excess imidazole was added to the solution prepared according to Example6 to dissociate the interaction between Ni-NTA and a histidine tag. Anequal volume of THF was added and stirred for 1 week to stabilize thehollow protein nanocage (10).

FIG. 14 shows the TEM photograph and schematic diagram (10) after theaddition of excess imidazole when eluting the inside polymers as in FIG.13. Here, it was confirmed that the addition of excess imidazoledissociated the binding between the polymer and protein, and thereby allthe residual polymers which had been bound to the protein-coated shellwere eluted out of the structure.

EXAMPLE 8 Effects of Solvents on Stability of Protein-Coated PolymerParticles and Protein Cage

The effects of solvents (DMF and THF) on the self-assembly of thepolymer-protein hybrid aggregates were investigated. Upon the additionof Ni-NTA-PS (Mn=21,800) in each solvent to the deionized water (pH 7.4)containing His6-GFP, the water-DMF system formed distinct sphericalaggregates, whereas water-THF formed indistinct large aggregates. It wasinferred to be due to the low solubility of His6-GFP in THF.

In order to prepare the polymer-protein hybrid aggregates, during theaddition of the polymer into the aqueous solution (pH 7.4) containingthe protein, 4 vol. % of DMF was added, which is a suitable solvent forNi-NTA-PS. The organic solvent DMF can exist in the core of thespherical aggregates, and can also exist outside the aggregates in theabove system. The presence of DMF in the core can cause the swelling ofpolystyrene. Therefore, in order to observe the effect of DMF on theaggregates, dynamically captured aggregates (the glass transitiontemperature of the polystyrene core is lower than that of these) wereformed, and DMF was removed from the system by dialysis after theformation of spherical aggregates. TEM measurements and a DLS studyshowed that the initial aggregates were maintained even after dialysis(24 h) (FIG. 15A). However, the precipitation of the aggregate occurredover time due to the phase separation. This result indicated that thepresence of DMF (4 vol. %) is essential in conserving thepolymer-protein hybrid aggregates in the aqueous solution. In addition,in order to confirm the change in the shape of polymer-proteinaggregates, an excess of an imidazole aqueous solution (250 mM) wasadded to the dialysis solution of the polymer-protein aggregates. TEMmeasurements and a DLS study showed that the size of polymer-proteinaggregates was decreased (24 h; FIG. 15B). It was inferred to be due tothe substitution of His-tagged GFP by the competitor, a ligandimidazole. After the addition of imidazole, the aggregates wereunstable, and grouped together over time to form indistinct and largeramorphous aggregates.

The possible mechanism for the in situ formation of the polymer-proteinhybrid aggregates inferred from the results of the experiments wasconfirmed to be due to the hydrophobic interaction of the internalNi-NTA-PS and the increased stability by the hybrid through theHis-tagged GFP. When Ni-NTA-PS dissolved in DMF was added to the aqueoussolution, aggregation began due to the hydrophobicity of polystyrene andthus-produced aggregates comprise a hydrophilic nickel-complexed NTAmoiety on the surface thereof, and the polystyrene matrix can therebyconstitute the reverse-micelle in the core, which can be stabilized bythe hydrophilic His-tagged protein through the NTA-Ni-histidineinteraction in the aqueous solution.

EXAMPLE 9 Size Control of Protein-Coated Polymer Particles

In order to investigate the effects of various factors controlling thesize of protein-coated polymer particles, the ratio of the amount ofpolymer to the amount of protein, pH of the reaction solution, themolecular weight of polymer, and the amounts used of the polymer andprotein were varied, and the size of the resulting aggregates wasmeasured to confirm the effects of these parameters on the size changeof the aggregates.

1. Effect of Polymer Concentration

Protein-coated polymer particles were prepared by a method similar tothat described in Example 1, wherein the ratio of the amount of polymerused to the amount of protein used was varied, and the size of theresulting particles was investigated.

As shown in FIG. 17, the results showed that the size of the resultingaggregates increased as the amount of polymer used to that of proteinincreased.

2. Effect of pH

Protein-coated polymer particles were prepared by a method similar tothat described in Example 1, wherein the pH of the solution was variedwithin the range of 6.5 to 8.5, and the size of the resulting particleswas investigated.

As shown in FIG. 18, the results showed that the size of the resultingparticles decreased as the pH of the reaction solution during thepreparation of aggregates increased.

3. Effects of Polymer Molecular Weight and Amounts of Polymer andProtein Used

The same experiment was performed as in Example 1, except that Ni-NTA-PSwith a molecular weight of 4,900 was utilized instead of Ni-NTA-PS witha molecular weight of 21,800 to prepare the aggregate together withHis6-GFP, and the shape and size were analyzed by DLS and a TEM imageand presented in FIG. 19 and Table 1. In addition, the size of theparticles prepared while reducing the amounts of the polymer and theprotein used was also disclosed in Table 1.

As shown in FIG. 19 and Table 1, taking into account that the size ofthe particles prepared using the high molecular weight polymer in thesame concentration is approximately 100 nm, when the molecular weight ofthe polymer used was decreased to 4,900, the size of particles formedwas confirmed to be remarkably increased to 280 nm to 350 nm.

In addition, as shown in Table 1, when the aggregates were formed whilereducing the concentrations of the polymer and protein in the sameratio, the size of particles formed was confirmed to be decreased as theamounts of polymer and protein used were decreased.

TABLE 1 ^(b)Mean diameter ^(c)Average size ^(a)Amount of Amount of His6-Rate of addition of micellar of micellar polymer in 0.1 GFP in 2.5 mL ofpolymeric aggregates aggregates Entry mL DMF [mg] H₂O [μg] solution[mL/h] (DLS) [nm] (TEM) [nm] 1 0.125 540 0.01 365 ± 25.48 ~350 2 0.0625270 0.01 342 ± 23.94 ~330 3 0.03125 135 0.01 103 ± 7.21  ~100 4 0.00312513.5 0.01 66 ± 4.62 ~50 5 0.001562 6.75 0.01 42 ± 2.94 ~50 ^(a)preparedby stepwise dilution from a higher concentration. ^(b)mean diameterobtained from number distribution DLS measurements.

PREPARATION EXAMPLE 2 Preparation of Polymer Comprising NHS FunctionalGroup

The reaction solution containing styrene (6.51 mL, 56.8 mmol) andanisole (3.5 mL) was deoxygenated by performing three cycles of afreeze-pump-thaw process. Then, CuBr (54.3 mg, 0.379 mmol), bpy (118 mg,0.757 mmol), and N-hydroxysuccinimidyl 2-bromo-2-methylpropionate (100mg, 0.379 mmol) were added to the reaction vessel, and pump-N₂substitution was repeated three times. The reaction solution was reactedat 110° C. for 10 h. The reaction solution was diluted with THF, and aneutral alumina column was used to remove the Cu catalyst. The solutiondeprived of the Cu catalyst was added dropwise to an excess of methanolto precipitate and purify the polystyrene having an NHS functional group(Mn: 12,000, PDI: 1.12).

EXAMPLE 10 Preparation of Protein-Coated Polymer Nanoparticles andProtein Nanocage by Covalent Bond 1. Preparation of GFP-Coated PolymerNanoparticles

Polystyrene having an NHS functional group (0.26 mg, 2.2×10⁵ mmol) wasdissolved in 0.4 mL of DMF, and this solution was added dropwise to aPBS buffer solution (10 mL, 50 mM, pH 8.0) containing His6-GFP (0.16 mg,1.5×10⁻⁵ mmol) at room temperature using a syringe pump at a rate of0.04 mL/h to prepare a structure. The shape and size of thethus-prepared structure were measured by dynamic light scattering (DLS)and TEM, and the results are presented in FIG. 20.

1.1. Preparation of GFP-Coated Nanocage

The process of removing the polymer core from the polymer nanoparticlescoated with GFP via a covalent bond as prepared in Example 10.1 wasfurther practiced to prepare the protein nanocage comprising GFP.Specifically, 1.0 mL of a 2.5% glutaraldehyde aqueous solution wasinjected into the GFP-coated polymer nanoparticles for 30 min. Next,sodium borohydride was added to stop the reaction. 5 mL of THF wasinjected into the reaction mixture, which was stirred for 12 h. Next,THF was removed and the polymer which was not bound to the protein wasremoved by a membrane syringe filter.

2. Preparation of RFP-Coated Polymer Nanoparticles

Polystyrene having an NHS functional group (0.26 mg, 2.2×10⁻⁵ mmol) wasdissolved in 0.4 mL of DMF, and this solution was added dropwise to aPBS buffer solution (10 mL, 50 mM, pH 8.0) containing His6-RFP (0.16 mg,1.5×10⁻⁵ mmol) at room temperature using a syringe pump at a rate of0.04 mL/h to prepare a structure. The shape and size of thethus-prepared structure were measured by DLS and TEM, and the resultsare presented in FIG. 21.

3. Preparation of YFP-Coated Polymer Nanoparticles

Polystyrene having an NHS functional group (0.26 mg, 2.2×10⁻⁵ mmol) wasdissolved in 0.4 mL of DMF, and this solution was added dropwise to aPBS buffer solution (10 mL, 50 mM, pH 8.0) containing His6-YFP (0.17 mg,1.5×10⁻⁵ mmol) at room temperature using a syringe pump at a rate of0.04 mL/h to prepare a structure. The shape and size of thethus-prepared structure were measured by DLS and TEM, and the resultsare presented in FIG. 22.

4. Preparation of Fibrinogen-Coated Polymer Nanoparticles

Polystyrene having an NHS functional group (0.26 mg, 2.2×10⁻⁵ mmol) wasdissolved in 0.4 mL of DMF, and this solution was added dropwise to aPBS buffer solution (10 mL, 50 mM, pH 8.0) containing fibrinogen (2.0mg, 1.5×10⁻⁵ mmol) at room temperature using a syringe pump at a rate of0.04 mL/h to prepare a structure. The shape and size of thethus-prepared structure were measured by DLS and TEM, and the resultsare presented in FIG. 23.

PREPARATION EXAMPLE 3 Preparation of Polymer Comprising BiotinFunctional Group 1. Synthesis of Biotinylated RAFT Reagent

Biotin (0.5 g, 2.0 mmol) and carbonyldiimidazole (0.64 g, 4.0 mmol) weredissolved in DMF (20 mL), and reacted for 6 h at room temperature.2-(2-Aminoethoxy)ethanol (0.63 mL, 6.0 mmol) was further added to thereaction solution and stirred for 18 h. After removal of the solvent,biotinyl alcohol was purified by column chromatography (stationaryphase: silica, mobile phase: 1-butanol/acetic acid/water=80/10/10).Purified biotinyl alcohol (0.35 g, 1.0 mmol) was dissolved in DMF andreacted with S-1-dodecyl-S′-(α,α′-dimethyl-α″-acetic acid)trithiocarbonate (0.37 g, 1.0 mmol), DCC (0.205 g, 1.0 mmol), and DMAP(0.015 g, 0.12 mmol) for 48 h at room temperature. After solidprecipitate filtration and solvent removal, the biotinylated RAFTreagent was purified by column chromatography (stationary phase:silica,mobile phase:chloroform/methanol=70/30).

2. Preparation of Polystyrene Functionalized by Biotin

Styrene (2 mL, 17.4 mmol), AIBN (1.43 mg, 0.01 mmol), and biotinylatedRAFT reagent (59.1 mg, 0.08 mmol) were added to anisole (0.8 mL)deprived of oxygen, and reacted at 65° C. for 131 h. Afterpolymerization, the reaction mixture solution was added to excessmethanol and precipitated to obtain the polystyrene having a biotinfunctional group (Mn: 9,200, Mw: 11,100, PDI: 1.18).

EXAMPLE 11 Preparation of Protein-Coated Polymer Nanoparticles andProtein Nanocage by Ligand-Receptor Binding

Polystyrene having a biotin functional group (0.2 mg, 2.2×10⁻⁵ mmol)prepared according to Preparation Example 3 was dissolved in 0.4 mL ofDMF, and this solution was added dropwise to a PBS buffer solution (10mL, 50 mM, pH 8.0) containing streptavidin (0.79 mg, 1.5×10⁻⁵ mmol) atroom temperature using a syringe pump at a rate of 0.04 mL/h to preparea structure.

EXAMPLE 12 Preparation of Protein-Coated Polymer NanoparticlesComprising Two or More Different Types of Proteins

Ni-NTA-PS (0.1 mg, 1.5×10⁻⁵ mmol) prepared according to PreparationExample 1 was dissolved in 0.2 mL of DMF, and this solution was addeddropwise to a PBS buffer solution (5 mL, 50 mM, pH 7.4) containingHis6-GFP (0.2 mg, 0.7×10⁻⁵ mmol) and His6-lipase (0.3 mg, 0.7×10⁻⁵ mmol)at room temperature using a syringe pump at a rate of 0.02 mL/h. Afteraddition over 10 h, the reaction mixture was stirred for one day.

PREPARATION EXAMPLE 4 Preparation of Polymer Bound Tri-NI-NTA at itsTerminal

As an example for the preparation of a polymer comprising threefunctional groups, which can bind to a protein, per one molecule ofpolymer, a polystyrene polymer which has three nickel-complexed NTAbound to its terminal was prepared. The method of preparing the polymeris shown in FIG. 24.

1. Synthesis of Protected Tri-NTA Initiator (6′)

Synthesis of Compound 1′

tert-Butyl bromoacetate (7.84 mL, 50.361 mmol) andN,N-diisopropylethylamine (DIPEA) (4.6 mL, 26.412 mmol) were added tothe suspension in which H-Lys(Z)-OtBu.HCl (2 g, 5.360 mmol) wasdissolved in 50 mL of DMF under a nitrogen atmosphere. The reactionmixture was stirred overnight at 55° C. Volatiles were evaporated at 65°C. under vacuum. A slurry residue was extracted with cyclohexane:ethylacetate (3:1). The extract was concentrated and analyzed bychromatography on silica gel using hexane:ethyl acetate (4:1) as amobile phase.

Yield: 2.8 g (92.5% on the basis of H-Lys(Z)-OtBu.HCl).

TLC: R_(f)=0.38 (hexane:ethyl acetate=4:1).

¹H NMR (300 MHz, CDCl₃), δ (TMS, ppm): 1.42 (s, 18H), 1.45 (s, 9H), 1.53(m, 4H), 1.62 (m, 2H), 3.20 (m, 2H), 3.31 (t, J=7.2, 1H), 3.44 (dd,J=16, 8.4, 4H), 5.08 (s, 2H), 7.34 (m, 5H).

Synthesis of Compound 2′

10% Pd/C (150 mg) was added to the methanol solution of 1′ (1.7 g/50 mL,3.013 mmol) under a nitrogen atmosphere. The reaction mixture wasstirred vigorously under a hydrogen atmosphere at room temperature for 9h. Pd/C was filtered on celite, and the filtrate was evaporated underreduced pressure.

Yield: 1.15 g (88.7% on the basis of 1′).

TLC: R_(f)=0.5 (chloroform:methanol=6:1).

¹H NMR (300 MHz, CDCl₃), δ (TMS, ppm): 1.44 (s, 18H), 1.45 (s, 9H), 1.63(m, 6H), 2.87 (t, J=7.2, 2H), 3.29 (t, J=7.6, 1H), 3.44 (dd, J=17.2,9.2, 4H).

Synthesis of Compound 3′

5.46 mL of trifluoroacetic acid (TFA) and 0.3 mL of triisopropylsilane(TIS) were added to the chloroform solution of 1′ (2 g/30 mL, 3.542mmol). The reaction mixture was stirred at room temperature and analyzedby TLC. After 3 h, 7 mL of methanol and 4 mL of water were added to thereaction mixture. Volatiles were evaporated under reduced pressure. Theresidue was dried azeotropically with toluene and precipitated fromanhydrous ethyl ether. The white precipitate was recovered and driedunder high vacuum.

Yield: 1.3 g (92.6% on the basis of 1′).

TLC: R_(f)=0.06 (chloroform:methanol:water=65:25:4).

¹H NMR (300 MHz, CDCl₃), δ (TMS, ppm): 1.48-1.53 (m, 4H), 1.66 (m, 1H),1.78 (m, 1H), 3.15 (m, 2H), 3.45 (t, J=12.8, 1H), 3.63 (dd, J=18, 9.6,4H), 5.08 (s, 2H), 7.36 (m, 5H).

Synthesis of Compound 4′

NHS (400 mg, 3.484 mmol), DMAP (58 mg, 0.468 mmol), and DCC (985 mg,4.772 mmol) were added to the solution in which 3′ (400 mg, 0.952 mmol)was dissolved in 20 mL of anhydrous DMF. The reaction mixture wasstirred at room temperature for 2 h, and to this mixture was added thesolution in which 2 (1.49 g, 3.479 mmol) and N,N-diisopropylethylamine(DIPEA, 0.63 mL, 3.479 mmol) were dissolved in 10 mL of chloroform.After reacting overnight, volatiles were evaporated at 65° C. undervacuum. The residue was dissolved in hexane:ethyl acetate (1:1), and thesolution was filtered to remove a urea slurry. Volatiles were extractedwith water three times. Combined extracts were concentrated and analyzedby chromatography on silica gel using chloroform:methanol (30:1) as amobile phase.

Yield: 1.23 g (74.6% on the basis of 3′).

TLC: R_(f)=0.34 (chloroform:methanol=30:1).

¹H NMR (300 MHz, CDCl₃), δ (TMS, ppm): 1.44 (s, 54H), 1.46 (s, 27H),1.53 (m, 16H), 1.63 (m, 8H), 3.20-3.31 (m, 12H), 3.44 (m, 16H), 5.08 (s,2H), 7.34 (m, 5H).

Synthesis of Compound 5′

10% Pd/C (130 mg) was added to the methanol solution of 4′ (1.0 g/40 mL,0.612 mmol) under a nitrogen atmosphere. The reaction mixture wasstirred vigorously under a hydrogen atmosphere at room temperature for 9h. Pd/C was filtered on celite, and the filtrate was evaporated underreduced pressure.

Yield: 0.8 g (87.1% on the basis of 4′).

TLC: R_(f)=0.48 (chloroform:methanol=9:1).

¹H NMR (300 MHz, CDCl₃), δ (TMS, ppm): 1.44 (s, 54H), 1.46 (s, 27H),1.53 (m, 16H), 1.63 (m, 8H), 3.20-3.31 (m, 12H), 3.44 (m, 16H).

Synthesis of Compound 6′

After the syntheses of Compounds 1′, 2′, 3′, 4′, and 5′ by theaforementioned methods, 2-bromo isobutyryl bromide (0.078 mL, 0.636mmol) and DIPEA (0.28 mL, 1.592 mmol) were slowly added to the dry THFsolution of 5 (0.8 g/15 mL, 0.533 mmol) under a nitrogen atmosphere at0° C. The reaction mixture was stirred overnight at room temperature.Volatiles were evaporated at 50° C. under vacuum. The residue slurry wasdissolved in dichloromethane and extracted with water three times.Combined extracts were concentrated and purified by flash columnchromatography on silica gel using chloroform:methanol (30:1) as amobile phase.

Yield: 0.63 g (71.7% on the basis of 5′).

TLC: R_(f)=0.26 (chloroform:methanol=30:1).

¹H NMR (300 MHz, CDCl₃), δ (TMS, ppm): 1.44 (s, 54H), 1.46 (s, 27H),1.53 (m, 16H), 1.63 (m, 8H), 1.88 (s, 6H), 3.20-3.31 (m, 12H), 3.44 (m,16H).

¹³C NMR (300 MHz, CDCl₃) δ (TMS, ppm): 23.30, 28.35, 28.43, 30.13,32.63, 34.40, 39.50, 48.77, 53.97, 65.20, 77.23, 170.08, 172.50.

As described above, in order to synthesize the polymer modified bymultivalent NTA at its terminal by atom transfer radical polymerization(ATRP), properly protected tri-NTA initiator (6′) was first prepared.First, at least two tert-butyl acetate groups were introduced to theα-nitrogen atom to convert H-Lys(Z)-OtBu.HCl to 1′. The selectiveremoval of the protecting group from 1′ provided the 1^(st) generationNTA dendron comprising one amino group (2′) or three carboxy groups(3′). Completely protected Compound 4′ was obtained by the coupling of2′ and 3′, and dendrimer 5′, which was modified at the amine group andprotected by tert-butyl, was obtained therefrom by catalytichydrogenation.

Thus-obtained 5′ was reacted with 2-bromoisobutyryl bromide to form anamide bond therebetween, thereby preparing ATRP initiator 6′ comprisingan activated alkyl bromide and an NTA moiety protected by tert-butyl atits terminal. In order to enhance solubility and block side reactions(e.g., proton addition reaction of ATRP ligand), a tert-butyl-protectedNTA-based amide initiator was designed. The structure of the synthesizedinitiator (6′) was confirmed by ¹H NMR and ¹³C NMR (FIG. 25). The peakat approximately 1.4 ppm in ¹H NMR and the peak at approximately 28 ppmin ¹³C NMR can be assigned as the tert-butyl proton. The molecularweight of 6′ was determined by gel permeation chromatography (GPC) andMALDI-TOF (matrix assisted laser desorption ionization-time of flight)mass spectrometry (FIG. 26). The molecular weight of 6′ by GPC was 1,650g/mol, and the m/z of the sodium adduct of the p-tri-NTA initiator byMALDI-TOF analysis was 1,672.772.

2. Preparation of Polystyrene Functionalized by Tri-NTA (8′)

Introduction of Tri-NTA into Polymer (7′)

Styrene (1.0 mL) and anisole (1.0 mL) were added to a Schlenk flaskfilled with nitrogen, three cycles of a freeze-pump-thaw process wererepeated, and then CuCl (17.5 mg) and dNbpy (71.27 mg) were added to theflask and two cycles of the freeze-pump-thaw process were furtherperformed. The flask was set up in an oil bath at 115° C., and thenp-tri-NTA initiator 6′ (140 mg, 85.2×10⁻³ mmol) was added and stirredfor 10 h. 0.1 mL aliquots were taken from the reaction mixture at timeintervals and diluted with THF for the GPC analysis. Precipitation frommethanol was performed to isolate the protected tri-NTA-bound polymer.

Removal of Protecting Group of 7′ (8′)

In a flask, the above-obtained protected tri-NTA-polystyrene(p-tri-NTA-PS) 7′ (100 mg, 15.63×10⁻³ mmol) was dissolved in 6.0 mL ofCH₂Cl₂. Trifluoroacetic acid (TFA, 0.96 mL, 14.06 mmol) was added to theabove flask. After completion of the addition of TFA, the reactionmixture was stirred at room temperature for 24 h. Finally, precipitationfrom methanol was performed to acquire the deprotectedtri-NTA-polystyrene (tri-NTA-PS, 8′).

Using the p-tri-NTA ATRP initiator (6′) prepared according toPreparation Example 4.1, the polymerization of styrene was performed at115° C. in solution phase (FIG. 24). Since amide-based initiatorsgenerally exhibit the initiation behavior in ATRP, halogen exchangetechnique was utilized to perform the polymerization reaction. In thehalogen exchange reaction, a CuCl/dNbpy catalyst was used together withthe initiator. Accordingly, 6′ and the CuCl/dNbpy catalyst were used tosynthesize p-tri-NTA polystyrene (p-tri-NTA-PS, 7′) by ATRP.

Gel permeation chromatography results showed that the molecular weightof the synthesized polymer increased over time, and there appeareddouble peaks due to the presence of excess initiator despite the use ofa halogen exchange technique (FIG. 27). However, after the polymerpurification by methanol precipitation, one symmetric elution peak wasobserved with narrow dispersion (Mn=6400 and

=1.15) (FIG. 27).

In the above-synthesized tri-NTA-bound polystyrene (7′), the presence ofan NTA moiety was confirmed by ¹H NMR (FIG. 28A) and ¹³C NMR (FIG. 29A).In FIG. 28, the peaks at 1.45 (a, (CH₃)₃—) and 1.46 ppm (b, (CH₃)₃—)were assigned as the tert-butyl proton, and the peaks at 2.8 (h, —CH₂—),3.2 (d, —CH—), 3.45 (c, —CH₂—), and 4.43 (m, —CH—Cl) ppm were assignedas the NTA moiety. From these results, 6′ was found to be successfullyused as the ATRP initiator. Polystyrene bound with tri-NTA at itsterminal (tri-NTA-PS, 8′) was prepared by removing the tert-butyl groupof 7′ with TFA in CH₂Cl₂. The structure of 8′ was also confirmed by ¹HNMR (FIG. 28B) and ¹³C NMR (FIG. 29B).

As described above, in the present invention, three NTA moieties wereintroduced at the α-chain terminal of polystyrene to obtain theamphiphilic linear-dendritic block copolymer (8′). Thus, the presentinventors have studied the self-assembly behavior of tri-NTA-PS (8′) inaqueous solutions. When water was slowly added to the THF solution oftri-NTA-PS (8′) in a glass vial under vigorous stirring at roomtemperature, self-assembled particles were formed. TEM and DLSmeasurements confirmed that these particles were spherical and uniformand had the diameter of approximately 40 nm to 60 nm (FIG. 30). Theseparticles can provide the surface with a hydrophilic NTA moiety, and canthereby be applied in various fields.

3. Preparation of Polymer Particles by Self-Assembly of Tri-NTA-PS

In a glass vial, tri-NTA-PS (8, 2 mg, Mn (GPC) of approx. 5,400) wasdissolved in 1 mL of dry THF. Next, 2 mL of water was slowly added undervigorous stirring at room temperature. After completion of the additionof water, the shape was investigated by TEM and DLS measurements whilecontinuously stirring the reaction solution.

EXAMPLE 13 Preparation of Polymer-Protein Hybrid Nanoparticles byBinding of his-Tagged Protein and Tri-NTA-PS

Polystyrene modified by nickel-complexed tri-NTA at its terminal(Ni-tri-NTA-PS, Mn of approx. 24,500, 0.25 mg, 1.02×10⁵ mmol) wasdissolved in 0.2 mL of DMF. In a glass vial, this polymer solution wasslowly added to 5 mL of deionized water comprising His6-GFP (27 kDa, 207g, 7.7×10⁶ mmol), using a syringe pump at a rate of 0.02 ml/h understirring at room temperature (18° C.). Several drops of PBS (50 mM, pHof approx. 7.4) were added to maintain the pH at 7.4 before the polymersolution was added to a water-soluble protein solution. After completingthe addition of the polymer solution (10 h), the resultingpolymer-protein hybrid solution was continuously stirred, during whichthe self-assembled form thereof was analyzed by DLS and TEM.

According to the aforementioned method for the preparation ofprotein-coated polymer nanoparticles, the self-assembled form oftri-NTA-PS (8′), which was conjugated with His6-GFP through theinteraction of NTA-Ni/His in water/DMF (DMF 4 vol. %) after beingcomplexed with nickel, was studied. When deionized water (5 mL, pH 7.4)comprising His6-GFP (27 kDa, 207 μg, 7.7×10⁻⁶ mmol) was added into thepolymer solution prepared by dissolving 0.25 mg of nickel-complexedtri-NTA-PS (Mn of approx. 24,500, 0.25 mg, 1.02×10⁻⁵ mmol) in DMF (0.2mL), spherical core-shell hybrid particles having the size ofapproximately 90 nm to 115 nm were obtained, as expected. DLS data andrepresentative TEM images were presented in FIG. 31, demonstrating theshape and size of the above particles. From the TEM image (right) ofFIG. 31, the outer layer of a particle could be confirmed, which wasconsidered as a protein layer. DLS data and TEM measurements showed thatthese hybrid particles of nickel-complexed tri-NTA-PS and His6-GFP werestable in water/DMF up to fifteen days, and they exhibited very highsimilarity to that prepared from Ni-NTA-PS.

As a result, the present invention confirmed the synthesis of tri-NTA-PSby ATRP and its binding to His6-GFP in water/DMF as well as theself-assembly of the polymer itself in water/THF. First, atert-butyl-protected NTA-based amide initiator was prepared andcharacterized with ¹H NMR, ¹³C NMR, GPC, and MALDI-TOF massspectrometry. The tert-butyl group was removed from the α-chain terminalof polystyrene to prepare polystyrene which had deprotected tri-NTAbound to its terminal (tri-NTA-PS). While tri-NTA-PS, due to itsamphiphilicity, self-assembled to form spherical particles having adiameter of approximately 40 nm to 60 nm in water/THF, nickel-complexedtri-NTA-PS and His6-GFP formed spherical core-shell hybrid particleshaving a diameter of approximately 90 nm to 115 nm in water/DMF throughthe interaction of NTA-Ni/His.

Since tri-NTA-PS comprises three NTA moieties, it is dendritic and moreamphiphilic. It suggests that the polymer-protein hybrid particles orprotein cage according to the present invention can be useful in similarapplications, and can particularly be applied in the production ofvarious self-assembled forms of the polymer itself, enclosure ofhydrophobic additives such as nanoparticles, dyes, etc., targeted drugdelivery, and protein purification.

The invention claimed is:
 1. A method for preparing a protein cage whichcomprises: a 1^(st) step of preparing an amphiphilic polymer comprisinga 1^(st) hydrophobic polymer and a 1^(st) hydrophilic functional group;a 2^(nd) step of preparing a hydrophilic protein comprising a 2^(nd)functional group binding to the 1^(st) functional group; a 3^(rd) stepof forming an amphiphilic polymer-protein hybrid by the binding of the1^(st) functional group and the 2^(nd) functional group, and formingcore-shell structured particles comprising a protein shell and anamphiphilic polymer core by the self-assembly of the amphiphilic polymerin a hydrophilic solvent; and a 4^(th) step of removing some or all ofthe hydrophobic polymer of the core part from the core-shell structuredparticles.
 2. The method of claim 1, wherein the protein retains itsactivity by maintaining its tertiary structure.
 3. The method of claim1, wherein the protein cage comprises one or at least two types ofproteins.
 4. The method of claim 1, wherein the method further comprisesa step of forming bindings between the shell-forming proteins by addinga cross-linking agent to the core-shell structured particles which areformed in the 3^(rd) step.
 5. The method of claim 1, wherein a 2^(nd)hydrophobic polymer not having the 1^(st) functional group is furtheradded during the 3^(rd) step.
 6. The method of claim 1, wherein anadditive is loaded in the protein cage.
 7. The method of claim 1,wherein the additive is added in the 3^(rd) step to be included in thecore part during the self-assembly or is injected into the protein cageformed in the fourth step.
 8. The method of claim 1, wherein removal ofsome or all of the hydrophobic polymer of the core part from thecore-shell structured particles in the fourth step is performed by theintroduction of (i) a competitor compound for the binding between the1^(st) and the 2^(nd) functional groups, or (ii) a compound thathydrolyzes the polymer moiety in the amphiphilic polymer-protein hybrid.